Bottom Line:
All 92 current smokers and 11 (28.7%) of 37 former smokers spontaneously expectorated sputum and also produced sputum when using the Lung Flute.The specimens were of low respiratory origin without contamination from other sources, eg, saliva.Therefore, sputum collected by the Lung Flute has comparable features as spontaneously expectorated sputum.

Affiliation: Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA.

ABSTRACTMolecular analysis of sputum can help diagnose lung cancer. We have demonstrated that Lung Flute can be used to collect sputum from individuals who cannot spontaneously expectorate sputum. The objective of this study is to further evaluate the performance of the Lung Flute by comparing the characteristics of parallel samples collected with and without the Lung Flute and the usefulness for diagnosis of lung cancer. Fifty-six early-stage lung cancer patients (40 current smokers and 16 former smokers) and 73 cancer-free individuals (52 current smokers and 21 former smokers) were instructed to spontaneously cough and use Lung Flute for sputum sampling. Sputum cytology and polymerase chain reaction analysis of three miRNAs (miRs-21, 31, and 210) were performed in the specimens. All 92 current smokers and 11 (28.7%) of 37 former smokers spontaneously expectorated sputum and also produced sputum when using the Lung Flute. Twenty-seven former smokers (70.3%) who could not spontaneously expectorate sputum, however, were able to produce sputum when using the Lung Flute. The specimens were of low respiratory origin without contamination from other sources, eg, saliva. There was no difference of sputum volume and cell populations, diagnostic efficiency of cytology, and analysis of the miRNAs in the specimens collected by the two approaches. Analysis of the sputum miRNAs produced 83.93% sensitivity and 87.67% specificity for identifying lung cancer. Therefore, sputum collected by the Lung Flute has comparable features as spontaneously expectorated sputum. Using the Lung Flute enables former smokers who cannot spontaneously expectorate to provide adequate sputum to improve sputum collection for lung cancer diagnosis.

f2-bmi-10-2015-055: The expression levels of the three miRNAs in sputum collected by the Lung Flute from 56 patients diagnosed with stage I NSCLC and 73 cancer-free smokers. Horizontal lines denote mean values. The three miRNAs (A–C) show statistical significance of expression levels between NSCLC patients and cancer-free smokers (all P < 0.01).

Mentions:
qRT-PCR for quantification of miRNA expression was successfully undertaken in the specimens, since all three miRNAs had ≤30 Ct values in each sputum sample. The three miRNAs displayed a significantly different level between NSCLC patients and control subjects (all P < 0.01) (Table 3) (Fig. 2). Furthermore, the individual genes displayed 0.77–0.85 AUCs (confidence interval [CI], 0.67–0.93) in the sputum samples collected by the Lung Flute (Table 3), and 0.78–0.82 AUCs (CI, 0.73–0.91) in the samples collected by spontaneous coughing for the identification of NSCLC (Supplementary Table 2) (all P > 0.05). The three miRNAs analyzed in combination had an AUC of 0.91 (CI, 0.84–0.97) in sputum collected by the Lung Flute (Table 3) and an AUC of 0.90 (CI, 0.82–0.96) in the samples collected by spontaneous coughing (Supplementary Table 2) (P > 0.05). The combination of the three miRNAs in the sputum samples by the Lung Flute produced a higher AUC compared with any single miRNA used alone (all P < 0.05) (Fig. 3). Subsequently, the three miRNAs used together created 83.93% sensitivity and 87.67% specificity in sputum collected by the Lung Flute for the identification of NSCLC (Table 3). Similarly, combined use of the three miRNAs in sputum collected by spontaneously coughing generated a higher AUC (0.90; CI, 0.82–0.96) compared with any single miRNA (0.73–83; CI, 0.73–0.91) (all P < 0.05). As a result, the panel of three miRNAs produced a sensitivity of 83.72% and a specificity of 87.50% for diagnosis of NSCLC in sputum collected by spontaneous coughing. In addition, Pearson’s coefficients showed a significant correlation between the molecular results generated from sputum samples collected by spontaneous coughing and sputum obtained by the Lung Flute (r = 0.849, P = 0.001). Therefore, there was no different performance of the sputum miRNA biomarkers for the diagnosis of NSCLC in the samples collected by the two approaches.

f2-bmi-10-2015-055: The expression levels of the three miRNAs in sputum collected by the Lung Flute from 56 patients diagnosed with stage I NSCLC and 73 cancer-free smokers. Horizontal lines denote mean values. The three miRNAs (A–C) show statistical significance of expression levels between NSCLC patients and cancer-free smokers (all P < 0.01).

Mentions:
qRT-PCR for quantification of miRNA expression was successfully undertaken in the specimens, since all three miRNAs had ≤30 Ct values in each sputum sample. The three miRNAs displayed a significantly different level between NSCLC patients and control subjects (all P < 0.01) (Table 3) (Fig. 2). Furthermore, the individual genes displayed 0.77–0.85 AUCs (confidence interval [CI], 0.67–0.93) in the sputum samples collected by the Lung Flute (Table 3), and 0.78–0.82 AUCs (CI, 0.73–0.91) in the samples collected by spontaneous coughing for the identification of NSCLC (Supplementary Table 2) (all P > 0.05). The three miRNAs analyzed in combination had an AUC of 0.91 (CI, 0.84–0.97) in sputum collected by the Lung Flute (Table 3) and an AUC of 0.90 (CI, 0.82–0.96) in the samples collected by spontaneous coughing (Supplementary Table 2) (P > 0.05). The combination of the three miRNAs in the sputum samples by the Lung Flute produced a higher AUC compared with any single miRNA used alone (all P < 0.05) (Fig. 3). Subsequently, the three miRNAs used together created 83.93% sensitivity and 87.67% specificity in sputum collected by the Lung Flute for the identification of NSCLC (Table 3). Similarly, combined use of the three miRNAs in sputum collected by spontaneously coughing generated a higher AUC (0.90; CI, 0.82–0.96) compared with any single miRNA (0.73–83; CI, 0.73–0.91) (all P < 0.05). As a result, the panel of three miRNAs produced a sensitivity of 83.72% and a specificity of 87.50% for diagnosis of NSCLC in sputum collected by spontaneous coughing. In addition, Pearson’s coefficients showed a significant correlation between the molecular results generated from sputum samples collected by spontaneous coughing and sputum obtained by the Lung Flute (r = 0.849, P = 0.001). Therefore, there was no different performance of the sputum miRNA biomarkers for the diagnosis of NSCLC in the samples collected by the two approaches.

Bottom Line:
All 92 current smokers and 11 (28.7%) of 37 former smokers spontaneously expectorated sputum and also produced sputum when using the Lung Flute.The specimens were of low respiratory origin without contamination from other sources, eg, saliva.Therefore, sputum collected by the Lung Flute has comparable features as spontaneously expectorated sputum.

Affiliation:
Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA.

ABSTRACTMolecular analysis of sputum can help diagnose lung cancer. We have demonstrated that Lung Flute can be used to collect sputum from individuals who cannot spontaneously expectorate sputum. The objective of this study is to further evaluate the performance of the Lung Flute by comparing the characteristics of parallel samples collected with and without the Lung Flute and the usefulness for diagnosis of lung cancer. Fifty-six early-stage lung cancer patients (40 current smokers and 16 former smokers) and 73 cancer-free individuals (52 current smokers and 21 former smokers) were instructed to spontaneously cough and use Lung Flute for sputum sampling. Sputum cytology and polymerase chain reaction analysis of three miRNAs (miRs-21, 31, and 210) were performed in the specimens. All 92 current smokers and 11 (28.7%) of 37 former smokers spontaneously expectorated sputum and also produced sputum when using the Lung Flute. Twenty-seven former smokers (70.3%) who could not spontaneously expectorate sputum, however, were able to produce sputum when using the Lung Flute. The specimens were of low respiratory origin without contamination from other sources, eg, saliva. There was no difference of sputum volume and cell populations, diagnostic efficiency of cytology, and analysis of the miRNAs in the specimens collected by the two approaches. Analysis of the sputum miRNAs produced 83.93% sensitivity and 87.67% specificity for identifying lung cancer. Therefore, sputum collected by the Lung Flute has comparable features as spontaneously expectorated sputum. Using the Lung Flute enables former smokers who cannot spontaneously expectorate to provide adequate sputum to improve sputum collection for lung cancer diagnosis.